Specific double-stranded probes for homogeneous detection of nucleic acid and their application methods

ABSTRACT

Double-stranded nucleic acid hybridization probes comprise a longer strand perfectly complementary to a preselected target sequence in an assay and a shorter second strand complementary to the longer strand. The strands are labeled with interactive labels such as a fluorophore and a quencher. The probes may be used in real-time amplification assays to distinguish among alleles.

[0001] This invention relates to novel probes for homogeneous andspecific detection, including real-time detection, of nucleic acids.

BACKGROUND OF THE INVENTION

[0002] Traditional heterogeneous detection methods for nucleic acidrequire separation of hybridized and urdlybridized probes, while newhomogeneous methods eliminate the separation steps, and are more rapid,simple and quantitative. A variety of nucleic acid amplificationtechniques have been developed and can amplify a specific sequence ofnucleic acid to several million copies within 2-3 hours. However, thedominant gel electrophoresis analysis greatly hindered their wideapplication in clinical diagnostics. Recently, the combination ofhomogeneous detection with these amplification techniques, especiallypolymerase chain reaction (PCR), greatly improved nucleic acid baseddiagnostics. The resulting quantitative real-time PCR assays arebecoming increasingly popular.

[0003] Current real-time fluorescence PCR assays can be classified intoprobe format and non-probe format. Probe format assays utilizefluorogenic probes, e.g. 5′-exonuclease (TaqMan™) probes, molecularbeacons, fluorescence energy transfer probes, Scorpion probes, light-upprobes, etc. Non-probe format assays utilize fluorogenic dyes, e.g. SYBRGreen I, to indicate the reaction. The non-probe format, though simple,finds rather limited application due to its inability to discriminatenon-specific amplification. In comparison, the probe format with asecond recognition step is much more reliable. However, culTent probesmentioned above are all difficult to design and synthesize, and they areexpensive. Another disadvantage of current probes is their limitedspecificity. Even molecular beacons, which are claimed to be the mostspecific ones, have to be modified to discriminate single-nucleotidemismatch in some cases.

[0004] This invention relates to a new probe that can be a homogeneousand specific probe for nucleic acid detection. This probe is based on aconcept different from the current probes. It is simple to design, easyto prepare, inexpensive, extremely specific, and can be combined withany current nucleic acid amplification technique.

[0005] Assays with double-stranded probes according to this inventionare based on competitive reaction between oligonucleotides rather thandirect hybridization as utilized in current probes. This new probe notonly can achieve in a much simpler way what the current probes can, butalso possesses many advantages over the current probes.

SUMMARY OF THE INVENTION

[0006] This invention relates to specially designed probes for nucleicacid detection and their applications. The probes can specificallydetect nucleic acid in a homogeneous format. The probes composed twocomplementary oligonucleotides of differing length that are labeled: onewith a fluorophore and the other with quencher or fluorescence acceptor.Under suitable conditions, the probes are double-stranded. When oneprobe strand hybridizes with target, the fluorophore generatesfluorescence change. Certain embodiments can specifically recognizetheir perfectly matched targets at room temperature, but cannot reactwith a “target” containing a single-mismatch. Probes according to thisinvention can be used for real-time nucleic acid amplification detectionassays.

[0007] Probes according to this invention can comprise DNA, RNA, ormixtures of the two. They can comprise non-natural nucleotides andnon-natural nucleotide linkages. Their 3′ ends may be blocked to preventextension. When we refer to “oligonucleotides” of the probes, we mean toinclude the foregoing.

[0008] This invention also relates to assays employing double-strandedprobes. Hybridization assays of this invention in which onlysingle-stranded target is present include probes as described above.Assays in which double-stranded target is present, such as typical PCRamplification, can include as well double-stranded probes havingcomplementary oligonucleotides of equal length.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 shows schematic drawing of a double-stranded probe and itsworking principle.

[0010]FIG. 2 shows schematic illustration of the working principle ofdouble-stranded probe in PCR detection during denaturation and annealingstages.

[0011]FIG. 3 shows reaction kinetics of double-stranded probe with itsperfectly matched and single-nucleotide mismatched target.

[0012]FIG. 4 shows real-time PCR detection with double-stranded probe.From up to down, templates are 10-fold serially diluted until the lastone that is water.

[0013]FIG. 5 shows single nucleotide mutation detection withdouble-stranded probes in real-time PCR utilizing in the same reactionvessel a double-stranded probe complementary to wild-type target and asecond double-stranded probe complementary to target with the mutation.

DETAILED DESCRIPTION OF THE INVENTION Composition of Double-strandedProbe

[0014] Double-stranded probes according to this invention are made oftwo complementary oligonucleotides of different lengths. One strand islabeled with a fluorophore and the other is labeled with a quencher. Inless preferred embodiments, the fluorescence quencher can be replaced bya fluorescence acceptor of the fluorophore. Double-stranded probes canhave different structures under different conditions, and this isreflected by the fluorescence change. When self-hybridized in a stabledouble-stranded structure, the fluorophore and the quencher, or thefluorescence energy donor and the acceptor, are in close proximity. Thefluorophore or the energy donor is quenched by the quencher or theenergy acceptor, and the probes are non-fluorescent at the emissionwavelength of the fluorophore or energy donor. When under denaturedconditions, such as in acid, basic or high temperature solution, the twostrands of the probe are separated, and the fluorophore (or energydonor) become fluorescent. In the presence of the target inhybridization solution, the longer strand of the probe can spontaneouslybind to the target, the double-stranded probe becomes dissociated, andthe fluorophore (or energy donor) become fluorescent.

Spontaneous Reaction Between Double-stranded Probes with Their Targets

[0015] Double-stranded probes having strands of different lengths canspontaneously react with single-stranded oligonucleotides in solution.In this reaction, the short strand in the double-stranded probe isdisplaced by the target oligonucleotide sequence to form athermodynamically more stable duplex. The resulting dissociation ofdouble-stranded probe produces an increase in fluorescence. In thisreaction, easily designed embodiments of the double-stranded probes havethe ability to distinguish perfectly matched targets fromsingle-nucleotide mismatched targets at room temperature. This extremelyhigh specificity lies in the fact that mismatched recognition isunfavored when compared with the self-reaction of the double strands ofthe probe itself. This is superior to single-stranded probes, becausesingle-stranded probe are thermodynamically unstable, and can behybridize with another single-stranded polynucleotide even there existsa mismatch. Molecular beacons are more specific than linear probes dueto their stable stem-loop structure that can out-compete a less stablemismatched reaction. However, the recognition portion of the molecularbeacons, the loop, is still single-stranded, and this leaves room formismatch hybridization, if the stem is not long enough or the loopsequence is too long. This is reflected by a recent report thatmolecular beacons cannot directly used for single-nucleotidediscrimination when combined with NASBA, a well-known isothermal nucleicacid amplification technique.

[0016] Referring to FIG. 1, double-stranded probe 1 is composed of twocomplementary oligonucleotides 2, 3 of different lengths. The longerstrand, in this case positive strand 2, is labeled with a fluorophore 4and the shorter negative strand 3 is labeled with a quencher 5. Theprobe is non-fluorescent due to the close proximity of the fluorophoreand the quencher. In the presence of target 6, negative strand 3 isdisplaced by the target, and the escaped fluorophore 4 becomesfluorescent. It will be appreciated that fluorescence would also result,if fluorophore 4 and quencher 5 are interchanged.

Combination of Double-stranded Probes with Nucleic Acid Amplification

[0017] As noted above, double-stranded probes according to thisinvention can spontaneously react with single-stranded target. We havediscovered that they also can be used to detect the newly producedsingle-stranded amplicon in a real-time format. During a typical PCRcycle comprising high-temperature denaturation, low-temperatureannealing and intermediate temperature elongation, double-strandedprobes are denatured during the denaturation (or melting) step, andbecome fluorescent. During the annealing step, in the absence of thetarget, the two strands of the probe will be in double-strandedconformation, and thus will be are non-fluorescent. In the presence ofthe target, however, the two probe strands will hybridize with thetarget. Fluorescence will be produced as a result. During the extensionstep, the probe strands move off the targets. By measuring thefluorescence during each annealing step of a PCR reaction, amplificationcan be tracked in a real-time format.

[0018] At the annealing stage this probe would undergo self-annealingand become non-fluorescent in the absence of target. However, in thepresence of the target, the fluorophore-labeled strand, say the positivestrand, of the probe would dissociate from the negative strand, bind tothe target, and become fluorescent. When the temperature is increased toallow extension of the primers (72° C.), the two strands of the probewould dissociate from the target and would not interfere with chainextension. By measuring fluorescence intensity during the annealingstage of every cycle, PCR can be followed in a real-time format.

[0019] Referring to FIG. 2, there is shown a double-stranded probe 21and a double-stranded amplicon 30, both of which are present in a PCRamplification reaction during an intermediate PCR cycle. Probe 21comprises strand 22, labeled with fluorophore 24, and complementarystrand 23, labeled with quencher 25. The labels are applied to theblunt-end termini of the strands. As depicted, strands 22, 23 are ofequal length, which they may be but need not be for use in real-timePCR. Amplicon 30 comprises complementary strands 31, 32. Uponhigh-temperature denaturation, strands 22, 23 of the probe separate, asdo strands 31, 32 of the amplicon. When the temperature is lowered tothe annealing temperature (for PCR primer annealing), probe strands 22,23 anneal, or hybridize, to their complementary target strands 31, 32 ofthe amplicon. Fluorophore 24 is not quenched by quencher 25, andfluoresces.

Design of Double-stranded Probes

[0020] The relative length of the two strands: in most cases, the twostrands of the probes are different in length, and usually, the longerstand is 1-5 bases longer than the shorter strand for PCR and 2-10,preferably 2-7, bases longer for isothermal allele discrimination. Incertain embodiments of assays according to this invention, such asdouble-stranded probes used in RNA detection or double-stranded probesused in real-time PCR, where both positive and negative target strandscompete to hybridize with probe strands, the two strands can be equal inlength.

[0021] The labeling position of the double-stranded probe: bothfluorophore and the quencher can be on the terminal or internal bases.In preferred embodiments, they are on opposed terminal complementarybases of the two strands. In especially preferred embodiments both thefluorophore and the quencher are on the blunt end of the probe. In somecases, especially when the probes are labeled with fluorescence energytransfer donor and acceptor, the position of the labels can be adjustedaccording the optimal energy transfer. In a preferred but not limitingembodiment, the fluorescence energy donor and acceptor are labeled onthe terminal bases of both strands, and one strand is usually blockedwith a phosphorate group.

[0022] Suitable instrument for double-stranded probes: double-strandedprobes can be combined with common nucleic acid amplification,especially PCR, and the amplicon can be measured in both real-time andend-point format. For real-time detection, fluorescence is measured atthe annealing temperature. Currently available real-timeamplification/detection instruments with which double-stranded probescan be used include the Model 7700 and Model 5700 from AppliedBiosystems (ABI), the IQ Cycler from Bio-Rad, the LightCycler fromHoffinaln-La Roche, and the Rotor-Gene 2000 from Corbett Research, amongothers.

[0023] The Advantages of double-stranded probes Simple and easy design:there are no additional requirements for the original reaction systemwhen designing double-stranded probes. Probe design itself is mucheasier compared with current dual-dye-labeled probes or adjacentlyhybridizing probes. Probes according to this invention can be designedby any persons who are familiar with conventional probe designs.

[0024] Cost effective preparation: the labeling procedure involved inpreparation of strands for double-stranded probes is only single-dyemodification, which can be carried out in any DNA synthesizer withoutadditional technical requirement. Purification involves only one step.This is much superior to other dual-dye-modification of probe strands orinternal modification of probe strands, where multiple step modificationand purification are needed, and the final yield is greatly reduced,thus increasing expense.

[0025] High specificity: it has already been proven by molecular beaconsthat structure-restricted probes possess higher specificity thanconventional linear probes. Double-stranded probes are a kind ofstructure-restricted probes in this context. The probes can bind totheir target only when the free energy produced is greater than that ofthe double-stranded probe. If there is mutation in the targets, thedouble-stranded probe may keep its own double-stranded state without anyreactions that are thermodynamically not favored.

EXAMPLE 1 Spontaneous Reaction a of Double-stranded Probe with itsTarget

[0026] Fifty μL of 0.80 μM double-stranded probe in 10 mM Tris-HCl (pH8.0) containing 1.5 mM MgCl₂ was maintained at 25° C., and itsfluorescence was monitored over time in an Eclipse spectrofluorometer(Varian). Fluorescence intensity was first measured for 2 minutes at 25°C. Then a two-fold molar excess (5 μL of 16 μM solution) of targetoligonucleotide was added, and the level of fluorescence was recorded at15-second intervals. The nucleotide sequences of the two probe strandswere 5′-FAM-ACGAACCTCAAACAGACACCAT-3′ (longer strand) and5′-TGTCTGTTTGAGGTTGCT-dabcyl-3′ (shorter strand). The targetcomplementary to the longer strand was 5′-CCATGGTGTCTGTTTGAGGTTGCT-3′,and the target containing a single-nucleotide substitution (mismatchedtarget) was 5′-CCATGGTGTCTGTTTCAGGTTGCT-3′, where an underlineidentifies the nucleotide substitution.

[0027]FIG. 3 shows the fluorescence (F) observed over time for both theperfectly complementary target (line 41) and for the mismatched target(line 42). It could be observed that over 20 times fluorescence could beachieved. If there is a single-nucleotide mismatch in the target, nofluorescence could be observed.

EXAMPLE 2 Real-time PCR Detection of Human β-globin with Double-strandedProbe

[0028] To test the utility of double-stranded probes as real-timeamplicon detectors in PCR assays, PCR amplifications were performed witha dilation series of target. Each 50 μL reaction contained 5 μL seriallydiluted template, 0.2 μM double-stranded probe, 0.4 μM of each primer,2.0 units of Taq polymerase, 200 μM of each deoxyribonucleosidetriphosphate, 50 mM KCl, 2.0 mM MgCl₂, and 10 mM Tris-HCl (pH 8.3).After denaturation at 94° C. for 5 min, 40 cycles of amplification (95°C. for 30 sec, 50° C. for 30 sec, and 72° C. for 1 min) were carried outin sealed tubes on a fluorometric thermal cycler (Rotor-Gene 2000,Corbett Research). Fluorescence was recorded at the annealing stage. Theoriginal extracted human DNA was serially diluted in tenfold steps andused as template. Water was used in place of the template for thecontrol sample. The double-stranded probe contains a nucleic acidsequence complementary to amplicons made from the target. For simplicitywe say that the probe is complementary to the target; however, personsfamiliar with amplification and detection will understand that by“target” we mean in this case both the original single-stranded targetand its complement, both of which are copied in exponential PCRamplification. A 268-base pair fragment of the human β-globin gene(GenBank code HuMMB5E, −195˜+73) was amplified. The forward and reverseprimers were 5′-GAAGAGCCAAGGACAGGTAC-3′ and 5′-CAACTTCATCCACGTTCACC-3′,respectively. The target sequence of the probe was located in the middleof the amplicon. The positive and negative probe strands were5′-FAM-AGCAACCTCAAACAGACACCATGG-PO₄-3′ and5′-GGTGTCTGTTTGAGGTTGCT-dabcyl-3′.

[0029] The results of real-time detection of fluorescence (F) measuredover forty cycles during PCR amplification are shown in FIG. 4. Initialtarget concentrations in the dilution series decrease from line 51, themost concentrated, to line 54, the least concentrated. Line 55 shows thenon-target (water) control.

[0030] When present in concentrations similar as the primers,double-stranded probes can react quickly with target strands. Thefraction of probes that do not find a target rapidly associate with eachother and quench their own fluorescence. Thus, they can be used inreal-time nucleic acid amplification assays. In PCR assays for thedetection of the human β-globin gene, we chose a negative strand of 20nucleotides with a melting temperature (Tm) close to that of theprimers, and a positive strand of 24 nucleotides in order to obtain aprobe-target hybrid that melted about 10° C. higher. We call this probethe “24/20 probe”. At the annealing stage this probe undergoesself-annealing and become non-fluorescent in the absence of target.However, in the presence of the target, the positive strand of the probedissociates from the negative strand, binds to the target, and becomefluorescent. When the temperature is increased to allow extension of theprimers (72° C.), the two strands of the probe dissociate from thetarget and do not interfere with chain extension.

[0031] Eleven double-stranded probes of different length (22/22 through22/17 and 20/20 through 20/16) were investigated, and they all workedwell in real-time PCR assays, even close in which both strands were thesame length. These observations demonstrated the great flexibility inthe design of double-stranded probes for real-time PCR.

EXAMPLE 3 Mutation Detection in Real-time PCR

[0032] To demonstrate the utility of probes according to this inventionin single-nucleotide mutation detection with real-time PCR, we preparedtwo DNA templates (targets) from the human β-globin gene that differedfrom one another by a single nucleotide substitution. We also prepared adouble-stranded probe complementary to the “wild-type” target and adouble-stranded probe complementary to the “mutant” target. We designedthe probes such that probe-target hybrids would melt about 10° C. higherthan a typical PCR annealing temperature (about 50° C.) and about 10° C.lower than the preferred extension temperature (about 72° C.) for TaqDNA polymerase. The probes were 24/20 probes (positive strand 24nucleotides in length, four nucleotides longer than the negativestrand). The probe complementary to wild-type target was labeled withFAM; the probe complementary to mutant target was labeled with TexasRed. Both had dabcyl quenchers. Both were blocked to prevent extensionof the probes. Each fluorophore could be distinguishably detected,because the two have different emission spectra. We ran four PCRreactions in which both double-stranded probes were present. Thereactions differed as to amplifiable target: none (Negative), wild-typetarget only (Wild), mutant target only (Mutant), and both wild-type andmutant targets (Wild+Mutant).

[0033] The positive strand of the probe specific to the wild-typesequence of human beta-globin gene was a 24-mer of the sequence:FAM-5′-AGCAACCTCAAACAGACACCATGG-3′-PO3 and the negative strand of theprobe was a 20-mer of the sequence: 5′-ATGGTGTCTGTTTGAGGTTGCT-3′-dabcyl.The two strands of the probe specific to the mutant version ofbeta-globin were: Texas Red-5′-AGCAACCTGAAACAGACACCATGG-3′-PO3 and5′-ATGGTGTCTGTTTCAGGTTGCT-3′-dabcyl. The two strands of each probe wereannealed with each other before adding them to the reaction mixture. TheDNA templates corresponding to the wild-type and mutant beta-globingenes were prepared by a in vitro mutagenesis method. The primers forthe real-time PCR were: 5′-GAAGAGCCAAGGACAGGTAC-3′ and5′-CAACTTCATCCACGTTCACC-3′. Each 50 micro liter reaction contained 5000copies of templates, 0.4 micro M primers, 0.2 micro M of positive strandand 0.24 micro M of negative strand of each probe, 4.0 mM MgC/2 alongwith other generic components required for PCR. After incubating thereaction mixtures at 94° C. for 5 min, 40 cycles of the thermal profile95° C. for 30 sec, 50° C. for 60 sec, and 72° C. for 1 min, were carriedout in a fluorometric thermal cycler. The fluorescence was monitoredduring the annealing steps. The results of real-time fluorescence versusPCR cycle number are shown in FIG. 5. Fluorescence from the probeagainst wild-type target is shown in filled circles (black dots).Fluorescence from the probe against mutant target is shown in unfilledcircles.

[0034] With the negative sample, there was no fluorescence (F) eitherfrom the wild-type probe, curve 71, or from the mutant probe, curve 72.The result showed that only when template is included in the reactiondoes one obtain an increase n fluorescence. With both targets present inthe sample, fluorescence increased markedly from both the wild-typeprobe, curve 73, and the mutant probe, curve 74. However, with wild-typetarget, fluorescence increased markedly from the wild-type probe, curve75, but not from the mutant probe, curve 76; and, conversely, withmutant target, fluorescence increased markedly from the mutant probe,curve 78, but not from the wild-type probe, curve 77. The results showedthat only the matched probe produced the right signal. Thediscrimination between wild-type template and mutant template wascomplete, 100%. This proved that probes according to this inventiondiscriminate between targets differing by a single nucleotide. Nosignals were observed when there were no templates, and two signals wereobserved when there were two templates.

[0035] We have investigated the temperature “window” in whichdouble-stranded probes are able to discriminate single nucleotidemutations. It has been shown in the literature that molecular beaconshave a larger window than linear probes and, thus, have betterdiscrimination. Nonetheless, the window for molecular beacons has beenshown not to be sufficiently large to permit discrimination at lowtemperatures, which explains the reported failure of molecular beaconsto discriminate such alleles in an isothermal amplification. We havefound that double-stranded probes according to this invention have evenlarger windows, which is believed to make them suitable fordiscrimination in isothermal amplification reactions.

We claim:
 1. A double-stranded nucleic acid hybridization probe for apreselected nucleic acid target sequence comprising a firstoligonucleotide comprising a first sequence perfectly complementary tosaid target sequence, a second oligonucleotide comprising a secondsequence that is complementary to said first sequence but is shorterthan said first sequence by up to ten nucleotides, a fluorophore labelattached to one of said first and second oligonucleotides, and a secondlabel from the group consisting of a quencher and a fluorescenceacceptor attached to the other of said first and second oligonucleotidesso as to interact with said fluorophore label when said oligonucleotidesare hybridized to each other.
 2. A double-stranded probe according toclaim 1 wherein said first and second oligonucleotides hybridize toproduce a double-stranded blunt end, and wherein said fluorophore labeland said second label are attached to said blunt end.
 3. Adouble-stranded probe according to either of claim 1 or claim 2, whereinsaid first and second oligonucleotides have 3′ ends that are blockedfrom being extendable by a polymerase.
 4. A double-stranded probeaccording to any of claims 1-3 wherein at least one of said first andsecond oligonucleotides comprises at least one non-natural nucleotide orat least one non-natural nucleotide linkage.
 5. In a real-time nucleicacid target sequence amplification and detection reaction in whichtarget amplicons are detected by a fluorescently labeled hybridizationprobe, the improvement comprising using as said hybridization probe adouble-stranded nucleic acid hybridization probe for said targetsequence comprising a first oligonucleotide comprising a first sequenceperfectly complementary to said target sequence, a secondoligonucleotide comprising a second sequence that is complementary tosaid first sequence, a fluorophore label attached to one of said firstand second oligonucleotides, and a second label from the groupconsisting of a quencher and a fluorescence acceptor attached to theother of said first and second oligonucleotides so as to interact withsaid fluorophore label when said oligonucleotides are hybridized to eachother.
 6. A real-time amplification reaction according to claim 5,wherein said sequence first and second oligonucleotides hybridize toproduce a double-stranded blunt end, and wherein said fluorophore labeland said second label are attached to said blunt end.
 7. A real-timeamplification reaction according to either of claim 5 or claim 6,wherein said first and second oligonucleotides have 3′ ends that areblocked from being extendable by a polymerase.
 8. A real-timeamplification reaction according to any of claims 5-7 wherein at leastone of said first and second oligonucleotides comprises at least onenon-natural nucleotide or at least one non-natural nucleotide linkage.9. A real-time amplification reaction according to any of claims 5-8wherein said second sequence is shorter than said first sequence by upto ten nucleotides.
 10. In real-time nucleic acid amplification anddetection reaction in which at least two target sequences are allelesdiffering by at least a single nucleotide, and in which amplicons foreach allelic target sequence are detected by a fluorescently labeledhybridization probe complementary thereto, the improvement wherein eachhybridization probe is a double-stranded nucleic acid hybridizationprobe for its allelic target sequence comprising a first oligonucleotidecomprising a first sequence perfectly complementary to said allelictarget sequence, a second oligonucleotide comprising a second sequencethat is complementary to said first sequence, a fluorophore labelattached to one of said first and second oligonucleotides, and aquencher label attached to the other of said first and secondoligonucleotides so that the fluorophore and quencher are in a quenchingrelationship when said oligonucleotides are hybridized to each other,and the fluorophore label of each of said double-stranded probes isdistinguishably detected.
 11. Real-time amplification reaction accordingto claim 10 wherein the second sequence of each double-stranded probe isshorter than the first sequence of that probe by up to ten nucleotides.12. A real-time amplification reaction according to either of claim 10or claim 11, wherein the first and second nucleotides of each probehybridize to produce a double-stranded blunt end, and wherein thefluorophore and quencher labels are attached to said blunt end.
 13. Areal-time amplification reaction according to any of claims 10-12wherein the first and second oligonucleotides of said probes have 3′ends that are blocked from being extended by a polymerase.
 14. Areal-time amplification reaction according to any of claims 10-13wherein at least one of the first and second oligonucleotides of atleast one of said probes comprises at least one non-natural nucleotideor at least one non-natural nucleotide linkage.
 15. An assay fordetecting a nucleic acid target sequence comprising adding to a samplesuspect to contain the target sequence a probe according to any ofclaims 1-4 and measuring a change in fluorescence.